PROJECT SUMMARY/ABSTRACT Sudden unexpected death in epilepsy (SUDEP) is the major cause of deaths associated with generalized seizures, and there are currently no effective therapies for this devastating disorder. Clinical observations and animal studies suggest that seizure-induced respiratory arrest (S-IRA) is the primary event leading to SUDEP. Although current studies implicate serotonin (5-HT) (also adenosine) in S-IRA, it is unclear if other neurotransmitters contribute to S-IRA, and how these neurotransmitters modulate S-IRA. These gaps in knowledge have significantly hindered the development of effective prevention strategies against SUDEP. Monoamine neurotransmitters in the brain, such as 5-HT, norepinephrine (NE) and dopamine (DA), are arousal-promoting and may stimulate breathing. In our preliminary and published studies in the DBA/1 mouse model, we determined that pharmacologic elevation of 5-HT or NE levels, and optogenetic stimulation of 5-HT- mediated arousal suppress S-IRA. Thus, in this high risk and high impact proposal, we hypothesize that enhanced monoamine-mediated arousal prevents seizure-induced sudden death in mice. We will test this hypothesis in two separate SUDEP mouse models, the DBA/1 mouse and the Dravet mouse that exhibits the salient features of Dravet syndrome (a type of epilepsy) in humans, including spontaneous seizures. In specific aim 1, we will determine if pharmacologic enhancement of monoamine function reduces S-IRA and/or sudden death in both DBA/1 and Dravet mice. We will examine if 5-HT, NE and DA are implicated in seizure- related mortality using monoamine reuptake inhibitors, and identify the specific serotonergic, adrenergic and dopaminergic receptors that are involved in prevention of seizure-related mortality. In specific aim 2, we will determine if selective stimulation of the dorsal raphe, locus coeruleus or ventral tegmental area reduces S-IRA in both DBA/1 and Dravet mice. We will use optogenetics to activate the monoaminergic brain nuclei that are known to mediate arousal response, and examine if enhancing arousal mediated by each monoamine suppresses S-IRA. If successful, the proposed studies could lead to significant conceptual advance in the mechanisms of SUDEP, and foster development of novel pharmacologic and neurostimulatory strategies against SUDEP. Completion of the proposed studies will establish animal models and methodology for further dissecting the neuronal circuitry, investigating the interaction, and elucidating the cellular mechanisms of monoamines involved in seizure-related mortality.